Microbial Control of Host Intercellular Communication

项目摘要/摘要 多细胞生物利用细胞间通讯来协调细胞功能和维持组织 动态平衡。最近的研究表明，这种交流在一定程度上是由细胞器的交换(通过 跨内吞作用)和直接跨越细胞-细胞连接的机械信号。这种交流的失调 会导致癌症和心血管疾病。尽管它很重要，但我们缺乏一个基本的分子 由于细胞生物学工具的数量有限，理解细胞间通信是如何发生的 能够探测细胞-细胞接触的分子机制。这项建议旨在澄清 被认为控制细胞间通讯的通路的调节机制通过研究它们是如何 在微生物控制下被操纵。单核细胞增多性李斯特氏菌通过人类传播 组织使用一种称为细胞间扩散的过程，这是一种囊泡介导的细胞间交换形式 模仿宿主的跨内吞作用。李斯特菌通过动员宿主的肌动蛋白细胞骨架在细胞间传播 用于细胞内运动和运输到细胞-细胞连接处。一旦到达交界处，它就会推动 并形成一个被相邻细胞吞噬的双膜突起。研究这一点 独特的传播过程将使我们能够研究几个悬而未决的细胞生物学问题。首先，是 细胞-细胞交界处用于吞噬微生物等大件货物的特定内吞途径？第二，是 机械敏感的膜域或膜曲率蛋白在李斯特菌推动下被激活 扩散过程中的交界处？为了回答这些问题，我们使用了高含量、基于图像的siRNA屏幕来 检测李斯特菌在传播过程中是否需要宿主细胞间的通讯途径。我们发现， 内吞和机械反应空泡蛋白CAV1、CAV2和PACSIN2促进李斯特氏菌的传播。我们 还揭示了其他19种宿主蛋白的假定作用，包括那些调节细胞膜曲率、反式- 内吞作用和粘连作用。我们的初步发现表明，总体假设李斯特菌颠覆了 促进细胞间传播的细胞间通讯途径。在目标1中，我们将确定PACSIN2如何 而小窝蛋白协调它们的活动，促进细胞间传播的吞噬阶段。在目标2中，我们将 揭示剩下的哪些特定地控制李斯特菌传播，它们是如何发挥作用的，以及它们是否起作用 单独地或与小窝一起。最终，我们提出的研究将改善我们的基础 了解宿主-微生物相互作用和基本细胞生物学，并可能揭示细胞间 在人类疾病中，沟通出了问题。

PROJECT SUMMARY/ABSTRACT Multicellular organisms use intercellular communication to coordinate cell function and maintain tissue homeostasis. Recent work suggests that this communication is driven in part by the exchange of organelles (via trans-endocytosis) and mechanical cues directly across cell-cell junctions. Dysregulation of this communication leads to cancer and cardiovascular diseases. Despite its importance, we lack a fundamental molecular understanding of how intercellular communication occurs because of the limited number of cell biological tools capable of probing the molecular mechanisms at cell-cell contacts. This proposal seeks to elucidate the regulatory mechanisms of the pathways thought to control intercellular communication by studying how they are manipulated when under microbial control. The bacterium Listeria monocytogenes disseminates through human tissues using a process called cell-to-cell spread, which is a vesicular-mediated form of intercellular exchange that mimics host trans-endocytosis. Listeria spreads from cell to cell by mobilizing the host’s actin cytoskeleton for intracellular motility and transport to the cell-cell junction. Once at the junction, it pushes against the membrane and forms a double-membrane protrusion that is engulfed by a neighboring cell. Studying this distinctive spreading process will allow us to examine several outstanding cell biological questions. First, are specific endocytic pathways used at cell-cell junctions to engulf large cargo like microbes? Second, are mechanically-sensitive membrane domains or membrane curvature proteins activated as Listeria pushes against the junction during spread? To answer these questions, we used a high-content, image-based siRNA screen to test if Listeria requires host intercellular communication pathways during spread. We discovered that the endocytic and mechanoresponsive caveolar proteins CAV1, CAV2, and PACSIN2 promote Listeria spread. We also revealed a putative role for 19 other host proteins, including those that regulate membrane curvature, trans- endocytosis, and adhesion. Our preliminary findings suggest the overall hypothesis that Listeria subverts multiple intercellular communication pathways to promote cell-to-cell spread. In Aim 1, we will determine how PACSIN2 and caveolins coordinate their activities to promote the engulfment stage of cell-to-cell spread. In Aim 2, we will reveal which of the remaining hits regulate Listeria spread specifically, how they function, and if they work independently or together with caveolae. In the end, our proposed studies will improve our fundamental understanding of host-microbe interactions and basic cell biology, and may uncover how intercellular communication goes awry in human disease.